The general subject matter of this invention, the microfluoroscope, is a device which dates to several publications in the 1940's and 1950's (Pattee, H. H., "The Microfluoroscope," Science, (1958) 128: 977-981). The microfluoroscope is essentially identical in principle to the common medical fluoroscope. In medical fluoroscopy, a patient is placed between a source of x-rays (an x-ray tube) and a fluorescent screen. The x-ray shadow of the patient's internal bones and organs are projected onto the fluorescent screen, converted to visible light, and viewed in real-time. Modern medical fluoroscopy has been improved over the years with the introduction of image intensifying devices which increase the visibility of the image, while lowering the x-ray dose to the patient.
A microfluoroscope is simply a fluoroscope in which the small fluorescent screen is viewed with an optical microscope, allowing the observation of object features too small to be seen with the naked eye. The microfluoroscope requires the use of extremely fine-grained or grainless fluorescent screens to prevent the image from being dominated by the structure of the phosphor itself. The phosphor layer is also preferably very thin, so that the light-emitting layer is completely within the depth of field of the optical microscope. The objects examined are generally thin specimens which are placed in direct contact, or very close proximity, to the phosphor layer. The phosphor is deposited onto a thin transparent substrate, which allows for the close approach of the high-aperture objective lens of an optical microscope from the opposite side of the substrate. Since very small objects are the subject matter of investigation with the microfluoroscope, extremely low energy (soft) x-rays are needed to achieve adequate contrast.
Microfluoroscopy is a type of contact x-ray microscopy, also known as microradiography. In standard contact microscopy, a sample is placed directly onto the surface of an x-ray sensitive recording medium. Originally, this medium was a fined grained silver halide photographic emulsion. After exposure, the medium is developed and the image examined using light microscopy. In certain cases, the silver grain structure of the developed emulsion can be prepared in a manner suitable for electron microscopy examination at higher resolution.
More recently, photographic emulsions have been replaced by x-ray sensitive photoresists which have a much smaller intrinsic structure (the polymer molecule size) than photographic emulsions. The exposure of the photoresist to x-rays causes radiation damage, which leads to variations in the solubility of the photoresist in a subsequent developer solution. Thus, the variable transmission of the x-rays through the specimen is translated into a relief image of the specimen on the photoresist surface. This image can be viewed at very high resolution using electron microscopy or atomic force microscopy. Specimen feature sizes near 100 .ANG. have been observed with this technique.
It is important to realize that the resolution of any contact microscopy scheme is limited by Fresnel diffraction. This resolution is given by: EQU .delta..apprxeq.(.lambda.d).sup.1/2
where .lambda. is the wavelength of the radiation and d is separation between the feature being imaged and the recording surface. Therefore, an extremely high resolution contact image is possible only for features very close to the recording surface. For example, with 25 .ANG. radiation, features 1 micron from the photoresist surface will be recorded at a resolution of no better than 500 .ANG..
There is a third type of contact microscopy that, like microfluoroscopy, is capable of real-time imaging. This microscope uses the photoconversion-contact method (Huang, L. Y., Z. Physik (1957) 149:225). In this technique, the specimen is placed on a thin x-ray transparent membrane. A photoemissive layer is deposited on the other side of the membrane, and this surface is in a vacuum. Photoelectrons are emitted into the vacuum by the photoemissive layer in response to the x-ray contact image of the specimen. These photoelectrons are accelerated, magnified by standard electron optics, and imaged onto an electron area-detector. An alternate scheme uses a simple point-projection principle instead of conventional electron optics (G. Hirsch, Point Projection Photoelectron Microscope with Hollow Needle, U.S. Pat. No. 4,829,177 (1989)). The photoconversion-contact method requires more complex and expensive instrumentation than a microfluoroscope.